For many years our technical support and scientific staff have been asked questions around the properties, use, and specific applications for Alexa Fluor® dyes. Here is a comprehensive set of frequently asked questions and their answers as put together by the Molecular Probes® technical support staff for the benefit of the researcher.

Antibodies, proteins, and peptides have primary amines and sulfhydryl groups that are readily labeled. Oligonucleotides are also often synthesized with either an amine or sulfhydryl group as a reactive "handle". Amine groups are the primary choice for labeling biomolecules, making proteins and antibodies among the easiest biomolecules to label. Life Technologies has developed a wide selection of amine-reactive moieties on our superior Alexa Fluor® dyes. Thiol-reactive reagents provide a means to modify a protein at a defined site with high specificity. Alexa Fluor® maleimides are excellent reagents for thiol-selective modification, quantitation, and analysis. The preferred bioconjugate should be highly fluorescent (i.e., have a high degree of labeling (DoL)), yet retain key functional parameters such as solubility, target affinity, and enzymatic activity.

Applications—Cell Analysis

At what concentration should an Alexa Fluor® IgG conjugate be used as a secondary antibody?

We recommend typical dilutions as follows:

Imaging applications—1:500 to 1:1000

Flow cytometry— 1:2000 or higher.

Because staining protocols vary with application, the appropriate dilution of antibody should be determined empirically. for the fluorophore- and biotin-labeled antibodies, including the phycoerythrin-, allophycocyanin-, and tandem-labeled antibodies, a final concentration of 1–10 μg/mL should be satisfactory for most immunohistochemical applications. for flow cytometry applications, 0.06–1.0 μg per 1 million cells should yield satisfactory results.

Do you offer a specific staining procedure (fix/permeabilization/block) for using Alexa Fluor® conjugates in immunocytochemistry (ICC) and immunohistochemistry (IHC)?

Although staining protocols may vary with application, the Alexa Fluor® conjugates can be used in place of any other secondary IgG conjugates that you are already using. They do not require a modification of your staining procedure.

Alexa Fluor® dyes are compatible with Triton®, formaldehyde, and formalin. Paraffin processing of tissues likely will extract dyes of almost any type, including Alexa Fluor® dyes, though they are fine to use after the processing, such as for immunohistochemistry (IHC) imaging.

Is Alexa Fluor® 405 the best dye for imaging at 405 nm?

The "best" dye really depends on the filter's emission specifications. Both Alexa Fluor® 405 dye and Alexa Fluor® 430 dye will efficiently excite at that wavelength.

Chemical Properties

How hydrophobic/hydrophilic are Alexa Fluor® dyes compared to other standard counterparts?

All of the Alexa Fluor® dyes have been engineered to be more hydrophilic than dyes such as fluorescein and Texas Red® dye. Alexa Fluor® 594 is the most hydrophobic of all of the Alexa Fluor® dyes.

What can quench Alexa Fluor® dyes?

Just like all dyes, Alexa Fluor® dyes are susceptible to typical quenchers such as chromogens (trypan blue), quenchers (QSY®), and other compounds such as 1, 4-diazabicyclooctane (DABCO) and N-propyl gallate (NPG), which, ironically, are also used as antifades.

What are the fluorescence quantum yields (QY) and lifetimes of the Alexa Fluor® dyes?

Performance

How are the Alexa Fluor® dyes better in performance than their standard counterparts such as fluorescein (FITC), Cy®3, Cy®5, Texas Red®, and allophycocyanin (APC) dyes?

Alexa Fluor® dyes make brighter and more photostable conjugates than do fluorescein (FITC), Cy®3, Cy®5, Texas Red®, or allophycocyanin (APC) dyes. Alexa Fluor® 488 vs. Fluorescein (FITC) or Cy®2 Fluorescein and Cy®2 conjugates rapidly quench as more fluorophores are added. The Alexa Fluor® 488 dye allows more fluorophores to be attached to the conjugate before self-quenching becomes apparent, leading to significantly brighter conjugates (Figure 1). This increased brightness means that you can use less conjugate in your experiments, reducing background fluorescence and stretching your research dollar, and enhanced sensitivity enables detection of low-abundance targets.

Figure 1. Comparison of the relative fluorescence of Alexa Fluor® 488 dye and FITC. Goat anti–mouse IgG conjugate fluorescence was determined by measuring the fluorescence quantum yield of the conjugated dye relative to that of a reference dye, and multiplying by the dye:protein labeling ratio.

Alexa Fluor® 555 vs. Cy®3 dye Alexa Fluor® 555 dye has higher photostability and quantum yield than tetramethylrhodamine isothiocyanate (TRITC) and rhodamine derivatives, which allow more time for observation and image capture. Alexa Fluor® 555 dye also has superior fluorescence output per protein or nucleic acid conjugate, surpassing that of any other spectrally similar fluorophore-labeled protein, including Cy®3–labeled proteins (Figures 2, 3). Other benefits include pH-insensitive fluorescence over a broad pH range, and solubility in water for easier labeling and detection.

Alexa Fluor® dyes have superior photostability In conclusion, the superior photostability of the Alexa Fluor® dyes allows more time for image observation and capture, thereby permitting greater sensitivity and simplifying the detection of low-abundance targets (Figure 7).

What antifade mountant do you recommend for cells stained with Alexa Fluor® conjugates?

For the best publication-quality imaging data, we recommend ProLong® Gold Antifade Reagent, which requires letting the sample cure at least overnight, which helps obtain crisper images. If there is a need to image samples without waiting overnight, we recommend SlowFade® Gold Antifade Reagent. For your convenience, these reagents come mixed with or without the nuclear stain 4', 6-diamidino-2-phenylindol (DAPI).

What is the brightest, most photostable, and best Alexa Fluor® dye?

All things being equal with the appropriate filters, Alexa Fluor® 488 is brighter and more photostable than other Alexa Fluor® dyes.

What are the two best Alexa Fluor® dyes?

If you have a typical fluorescence microscope with a BGR filter and no other constraints, then Alexa Fluor® 488 and Alexa Fluor® 594 are good choices, as the blue dyes generally have lower intensity.

Do I need to buy new emission filters to spectrally match the Alexa Fluor® dyes?

No, you do not have to buy special filters for Alexa Fluor® dyes, as these dyes have nearly identical spectral properties and generally better quantum yield compared to standard dyes. You can use the same filters that you use for the standard dyes (Table 1).